The external control of the expression of selected gene products in plants through the application of chemical substances can provide important agronomic and foodstuff benefits. The ability to externally control the expression of introduced genes in transgenic plants could be used to (1) prolong or extend the accumulation of desirable nutritional food reserve in seeds, roots, or tubers, (2) produce and accumulate products in plant tissues that are convenient for harvest and isolation, and (3) initiate expression of a toxin at the site of pathogen attack, possibly avoiding contamination of the ultimate food product with the toxin. These and other benefits have been unattainable in the field since means to bring known plant genes under external control in the field have not been available.
While technology exists to transform plants with the genes for selected products, their expression is either continuous throughout the lift cycle (controlled by a constitutive promoter), or is regulated by the developmentally timed program of maturation inherent in each organ/tissue/cell (stage or tissue specific promoters). Continuous expression precludes production at particular stages, in specific tissues or in response to environmentally unpredictable events. Such expression could place a major penalty on yield, due to greatly increased energy demands accompanying prolonged synthesis of the product. Tissue or stage specific expression, although valuable for the temporal and spatial accumulation of products, is under the variable timing of the developmental program of the plant. Precise developmental control of gene expression would necessitate the isolation of a multitude of stage and tissue specific promoters in all crop plants of interest. It wold be desirable to externally control the expression of an introduced gene product by the application of a specific chemical which can induce a specific promoter to activate the accumulation of a desired gene product. Hence, one chemical/promoter combination can be interactive at any stage or in any tissue available to the chemical, throughout the life cycle of any one of a large number of transformable plants, to control any desired gene product.
Certain genes can be induced by environmental factors such as light, heat shock and anaerobiosis. The promoters of these inducible genes from plants have been extensively analyzed (c.f., Kuhlemeier et al., Ann. Rev. Pl. Physiol., 38:221-257 (1987)). However, the inducers of these genes (i.e., light, temperature and O.sub.2 levels) are not easily or practically controllable under normal agronomic techniques.
Numerous chemicals, both natural and synthetic, have been shown to affect the growth and development of plants. A number of these plant growth regulators occur naturally, such as the hormones auxin, gibberellic acid, cytokinins, ethylene and abscisic acid (c.f., Davies, P. (Ed.) Plant Hormones and Their Roles In Plant Growth and Development, Martinus Nijhoff Publ. (1987)), while others have equally dramatic effects such as salicylic acid (Hooft Vanhuijsduijnen et al., J. Gen. Virol., 67:235-2143 (1986)). When these growth regulators are applied to various plant cells/tissues/organs, a change in the metabolism of the plant is observed which has been shown with each of the above regulators to result from new gene expression. Some products of these genes, as well as some genes themselves, have been isolated and characterized. The instant invention focuses on the phytohormone abscisic acid (ABA), 3-methyl-5-(1'-hydroxy-4'-oxo-2',6',6',-trimethyl-2'-cyclohexene-1'-yl)-ci s, trans-2,4-pentadienoic acid (c.f. Addicott, Abscisic Acid, Praeger Publ., N.Y. pp. 607 (1983)). ABA is involved in numerous processes such as stomatal closure, bud/seed dormancy, inhibition of seed germination, as well as in responses of plants to physical perturbations or stresses such as temperature and water (c.f. Zeevaart and Creelman, Ann. Rev. Plant Physiol., 39:439-473 (1988)). Numerous compounds have been described that mimic ABA in these physiological process, i.e., they are ABA-like (c.f. Walton, In Abscisic Acid (Ed. Addicott), Chapt. 4, pp. 113-146 (1983)). Our immediate interest focuses on the involvement of ABA in two of these key plant processes; accumulation of storage reserves in seeds and the response of plants to water stress (drought).
During seed formation in a wide variety of seeds, the endogenous ABA level increases dramatically, as it does in water-stressed tissue. Genes from the seeds of six plants (cotton, soybean, rice, maize, wheat and rapeseed) have been isolated that respond to ABA (when tissue is treated in vitro with the phytohormone) by accumulating the products of these same genes. The response is an increase in the mRNA levels of these specific genes (c.f., Quatrano, Oxford Surveys Pl. Mo. Cell. Biol., 3:467-477 (1986)). Although the characterization of some of these genes has been extensive, only in the case of the .beta.-conglycinin gene from soybean has an ABA-regulated gene been expressed in a transgenic plant; in petunia and tobacco by Bray et al., Planta 172:364-370 (1987) and in tobacco by Barker et al., Proc. Natl. Acad. Sci. (USA) 85:458-462 (1988).
The goal in each case is to identify the DNA sequences that control the expression of these genes in a tissue-specific, stage-specific and environmentally regulated manner. Bray et al. demonstrated this specific regulation in transgenic plants when a 4.2 kilobase (kb) fragment of the natural gene was transferred. In addition to the coding region, it contained a 1.1 kb 5' fragment and a 1.3 kb 3' fragment. Barker et al. transferred to tobacco a much larger fragment (12.3 kb) that contained more genes that the .beta.-conglycinin gene. Neither group characterized further, which part(s) of these large fragments might be responsible for the specific ABA induction effect, nor did they report treating transformed tissue with ABA to determine if the foreign gene would response to hormone addition.
Gomez et al., Nature 334:262-264 (1988), reported that when immature maize embryos, like wheat, are cultured in the presence of ABA they express a new set of genes in response to the hormone. One ABA-regulated gene (pMAH9), isolated in this report, codes for a 15.4 Kd protein. The mRNA for this protein is also expressed when mature leaf tissue is wounded or water stressed. The complete sequence of the coding region and parts of the 3' and 5' flanking regions are given, but no information is reported as to the sequence within the promoter region which is essentially for ABA regulation.
Mundy and Chua, EMBO Journal, 7:2279-2286 (1988), characterized the full genomic sequence of a gene from rice (RAB 21) which is induced by ABA, as well as by salt (NaCl), and water stress in numerous tissues, e.g., roots, leaves, embryos, suspension cultures. Although the complete sequence of the coding region (approximately 1 kb) and the 5' regulatory or promoter region (approximately 1.5 kb) is given, no information is reported as to the sequences within the promoter region which are essential for ABA regulation of the coding sequences. There is no teaching regarding the isolation of a promoter fragment, or its potential utility to achieve external control of plant gene expression. The authors themselves (p. 2285) state that "the regulatory roles of these different GC-rich repeats remains to be established. . . . " Applicant's invention lies in the isolation of promoter fragments, their use in constructs to transform protoplasts and plants, and in achieving the external control of plant gene expression.
Other seed-specific genes that have not been shown to be regulated by ABA, or by any other hormone, have been analyzed in the same manner. For examples, Chen et al., Proc. Natl. Acad. Sci. (USA) 83:8560-8564 (1986) showed that the region between -159 and -257 base pairs (bp) 5' to the transcription start site of the .alpha.-conglycinin gene is the required region for seed specificity. More recently, Chen et al., EMBO Journal, 7:297-302 (1988), constructed a chimeric gene comprising a 170 bp fragment from the .alpha.-conglycinin promoter region (-78 to -257) in different positions and orientations to the constitutive viral 35S promoter linked to the reporter gene chloramphenical acetyl transferase (CAT). They showed that the 170 bp 5' fragment enhances expression of the CAT gene in a tissue-specific and temporally regulated manner.
However, delineation of the specific regulatory regions required these investigators to assay for expression in transgenic plants at the stage of seed formation. This required long periods of time until the plants flowered and set seed (several months). Transient assays for promoter analysis have been reported and represent a faster way to delineate those promoter fragments that are regulatory. However, none of these reports which follow involve the use of a chemical inducer.
Ebert et al., Proc. Natl. Acad. Sci. (USA) 84:5745-5749 (1987), disclose studies of the active fragment of DNA constituting the nopaline synthase promoter. This promoter is constitutive rather than inducible, and while of bacterial origin, operates in a wide range of plant tissues. A construction was made such that the promoter controlled the expression of the reporter gene CAT. The authors reported that a fragment of 33 bp (-97 to -130) of DNA was necessary to promote expression of the CAT gene. They reported further that the presence of two copies of the fragment tripled the expression of the CAT gene. These results from stably transformed tobacco tissue were repeatable in a transient assay using tobacco protoplasts. Comparison of the level of CAT gene expression controlled by the fragments in both the transient expression of tobacco protoplasts and stably transformed tissues resulted in some differences. The authors nevertheless indicated their belief that such transient assays are valuable for "the study of upstream elements and trans-acting factors".
Howard et al., Planta, 170:535-540 (1987), studied the anaerobic induction of the maize alcohol dehydrogenase (Adhl) gene by electroporating gene fragments of Adhl into maize protoplasts from suspension culture cells, subjecting these protoplasts to reduced oxygen levels and assaying for Adhl expression 20 hours later. To facilitate measurement of Adhl gene expression regulated by anaerobiosis, they fused the 5' promoter or regulatory fragment of the native Adhl gene (1096 base pairs) to CAT. Their results demonstrated the normal regulation of an inducible promoter from a monocot maize gene (i.e., Adhl) when electroporated into protoplasts derived from a homologous cell culture system. They showed that just the Adhl promoter fragment, without the coding and 3' regions of the Adhl gene, is sufficient for anaerobic induction of the CAT gene.
Lee et al., Plant Physiology 85:327-330 (1987), have reported further definition of the size of the DNA fragment responsible for anaerobic induction of the maize Adhl gene. Utilizing a plasmid vector that facilitated placement of any desired gene adjacent and 3' to the promoter, they inserted the gene for CAT next to the Adhl gene. Using this plasmid, they transformed maize protoplasts and measured the production of CAT 24 hours later. By altering the size of the promoter DNA sequences in the construction, Lee et al. determined that 146 bp 5' to the transcription start site were sufficient to place the production of CAT under anaerobic induction. However, the expression of CAT was increased 5.times. or 8.times. by the addition of 266 or 955 bp, respectively, of contiguous 5' promoter sequences.
Walker et al., Proc. Natl. Acad. Sci (USA) 84:6624-6628 (1987), continued the studies of the DNA sequences of the 5' promoter region of the maize Adhl gene required for gene expression induced by anaerobic conditions in a transient assay. They determined that control of anaerobic induction of gene expression resided in two sequences from the promoter: those being the sequence between -133 and -124 bp and the sequence between -113 and -99 bp (5' to the transcription start site). Both sequences are necessary. Attachment of this 40 bp element to an unrelated viral promoter confers anaerobic regulation.
Ellis et al., EMBO Journal 6:11-16 (1987), indicate that when the fragment of DNA between base pairs -1094 and +106 bp of the maize Adhl gene was placed next to the sequence of DNA encoding CAT and the construction was stably transformed into tobacco cells, only extremely low levels of CAT gene expression could be observed under appropriate anaerobic conditions. In fact, only CAT messenger RNA was detected. However, promoter elements from the octopine synthase gene of bacteria, or those from the Cauliflower Mosaic Virus (CaMV), linked 5' to the Adhl promoter, stimulated the expression of the CAT gene and permitted detection of CAT after anaerobic induction. The fragment of DNA, consisting of 247 bp adjacent and 5' to the transcription start site of the structural gene for Adhl, was sufficient to put the expression of the CAT gene under anaerobic control. Therefore, anaerobic control by the 247 bp fragment of DNA was maintained even when the octopine synthase and CaMV 35S promoters, which are constitutive promoters, were present. The region of the Adhl promoter responsible for anaerobic induction demonstrated in transient assays by Howard et al., Lee et al., and Walker et al. were similar to the region showing anaerobic induction in stably transformed plants by Ellis et al.
Patents have been issued to animal and microbial systems in which selected gene sequences have been induced by chemicals in the environment that interact with certain regulatory sequences. U.S. Pat. No. 4,579,821, issued to Palmiter and Brinster, discloses the isolation of promoter/regulator sequences of the mouse metallothionein-I gene and its use to control the expression of selected DNA sequences operably linked to the promoter by exposure to heavy metal ions or steroid hormones. The expression of thymidine kinase fused to the metallothionein-I promoter was obtained in differentiated cells of adult mice upon administration of cadium or dexamethasone. U.S. Pat. No. 4,703,005, issued to Nakata and Shinagaua, discloses the isolation of a gene for phosphate-binding protein (phoS) to which was fused a foreign gene 3' to phoS. The foreign gene is controlled by phosphate in the culture medium.
Despite considerable effort to characterize the molecular basis of the response of plant tissue to hormones, the signal/transduction pathway from chemical to gene is not well understood. While reports of plant promoter sequences stimulated by light and anaerobic stress have appeared, no disclosures of inducible plant promoters responsive to chemical substances which can be applied in the field to control gene expression have appeared. At this time, a clear need exists for promoter sequences and recombinant constructs for the transformation of plants which would enable external control of selected genes which can confer agronomic advantages. Further, this specificity of expression should be amenable to external control through exposure to chemical substances which can be readily manipulated.